Project Summary The objective of our proposed study is to continue to improve the accuracy and usability of our prototype NanoDisk-MS assay for the detection of tuberculosis (TB) in blood samples. In Aim 1 we will add previously identified Mycobacterium tuberculosis (Mtb)-specific tryptic peptides of Ag85B to our prototype assay to improve sensitivity and explore the optimal number of peptides for antigens CFP-10, ESAT-6 and Ag85B required to maximize specificity of the test using previously-collected blood samples from 1,500 patients with and without TB (samples from the NIH-funded Clinical Suspicion of Tuberculosis study (CSTB) R01AI053731). In Aim 2 we will validate the performance of the optimized assay using previously collected blood samples from 250 patients and compare its performance on a standard MALDI-TOF MS to the Mini-12 instrument (samples from the NIH funded Global Consortium for Drug- resistant TB Diagnostics [GCDD U01AI082229]). In Aim 3 we will evaluate final assay performance using blood samples from 3,000 prospectively enrolled patients at risk for TB in clinics in Moldova, Mexico and the US to determine how well the assay distinguishes patients with active TB disease from those with latent TB, non-TB pulmonary diseases, and healthy controls. Our goal is to improve the sensitivity and specificity of our prototype assay in order to meet World Health Organization (WHO) performance criteria for TB screening (sensitivity:95%, specificity:90% ) and diagnosis (sensitivity:98%, specificity:98% ).